P < 0.05). We suggest that these different pathways work together via protein signaling and result in the pathogenesis and progression of MN.Membranous nephropathy (MN) is an autoimmune inflammatory disease in which proteins related with plenty of biological processes play an important role. However, the role of these proteins in the pathogenesis of MN is still unclear. This study aimed to screen differential proteins in kidney tissue samples from MN patients by using protein arrays and determine the pathways involved in the pathogenesis of MN. This study first tested a quantitative protein array (QAH-INF-3) and two semiquantitative protein arrays (L-493 and L-507) with normal renal tissue and identified L-493 as the most appropriate assay to compare protein levels between MN tissues and normal control tissues. The L-493 array identified 66 differentially expressed proteins (DEPs) that may be associated with MN. The gene oncology (GO) and protein-protein interaction (PPI) analyses revealed several processes potentially involved in MN, including extracellular matrix disassembly and organization, cell adhesion, cell-cell signaling, cellular protein metabolic process, and immune response ( Membranous nephropathy (MN) is an autoimmune inflammatory disease and the primary cause of nephrotic syndrome in adults, contributing to 20%–40% of end stage renal disease (ESRD) cases . The chaAccording to the previous study, autoantibodies and complements play important roles in the development and progression of MN . In 2009Protein microarrays are advanced and efficient technology to study proteins involved in kidney disease systematically because the arrays have the potential to screen thousands of proteins in a high-throughput manner with a smaller sample as compared with mass spectrometry (MS) and other traditional methods such as western blot and enzyme-linked immunosorbent assays (ELISAs) , 15. AccOur study was approved by the institutional research ethics committee of the Chinese People's Liberation Army General Hospital (No. S2015-061-01). All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and national research committee and with either the 1964 Declaration of Helsinki and its later amendments or the comparable ethical standards. All of the samples were obtained from the Nephrology Department of the Chinese People's Liberation Army General Hospital. A total of 15 kidney samples harvested via percutaneous renal biopsy and nephrectomy were included in the study as a discovery set: six samples from idiopathic MN patients and nine normal samples from renal cell carcinoma (RCC) patients. All of the MN patients are IMN diagnosed explicitly by renal biopsy pathology with exceptions for other diseases such as autoimmune diseases, infection, diabetes, and hypertension. There are 25, 28, and 32 glomeruli in each quality control specimen, respectively.μL.After the fresh samples were collected, the impurities and blood were washed out with PBS immediately. The process of centrifugation and removal of the supernatant was repeated for the samples until the tissue became nearly colorless. The tissue was dissociated and lysed according to the manufacturer's instructions. After the lysates were centrifuged, the protein concentrations in the supernatant were detected by using BCA methods . The samples were diluted in sample buffer to the desired concentration in a volume of 100 μg and 6.25 μg samples from a single tissue were used for QAH-INF-3, and 50 μg and 25 μg from the same kidney sample were used for L-507 and L-493.We chose the three following protein arrays to compare their performance in assessing the protein levels in kidney tissue: the quantitative protein chip QAH-INF-3  and the semiquantitative protein L-series antibody array chips L-507 and L-493 . QAH-INF-3 is a type of multiplexed sandwich ELISA-based quantitative array platform that enables researchers to accurately and simultaneously detect the concentration of 40 proteins by building a standard curve with a serial dilution of standard samples. L-507 and L-493 could separately detect 507 and 493 proteins based on biotin labeling and laser fluorescence scanning techniques, respectively. For detection, 12.5 Protein hybridization was conducted with the QAH-INF-3, L-507, and L-493 arrays according to the respective manufacturer's instructions. The fluorescent images were scanned by an Axon GenePix , and the data were analyzed with GenePix Pro 6.0 software .Hybridization proceeded according to the manufacture of QAH-INF-3, L-507, and L-493. The signal fluorescence images were scanned by Axon GenePix . The data was analyzed with software GenePix Pro 6.0 .To verify the results, EpCAM-1, CTSD , and ADAMTS-4  were selected as confirmation markers and tissue lysates of MN and N were measured by ELISA kits. The procedures were carried out following the specification of the kits.To avoid the false overexpression of transmembrane proteins caused by tissue lysates, we performed immunohistochemistry of MN and normal control with CTSD and CD46. Immunohistochemistry was carried out following the conventional methods of fixing tissues, paraffin-embedding, serial section (3 mm), antigen retrieval, blocking endogenous peroxidase activity, adding antibodies, incubation, and microscopic examination. The antibodies of CTSD and CD46 are both mouse monoclonal antibody .μg versus 6.25 μg group in QAH-INF-3, 50 μg versus 25 μg group in L-507 and L-493; the theoretical ratio was 2.0) as measurable proteins in kidney tissue and counted the number of available proteins on each chip to assess the performance of protein chips.The protein array performances among QAH-INF-3, L-507, and L-493 were assessed by the fold change of protein expression between the two concentrations of the loading samples. We defined proteins with a ratio between 1.3 and 3.0 (12.5 t-test (t-test) was used to address the significance of the quantitative data. Proteins with a fold change of MN/N > 1.4 and a P value < 0.05 were considered to be differentially expressed proteins (DEPs). The Database for Annotation, Visualization and Integrated Discovery v6.8 Beta  was used for gene oncology (GO) analysis. The Search Tool for the Retrieval of Interacting Genes/Proteins v10  was applied for protein-protein interaction analysis.Student's P > 0.05) while the urine protein and albumin (Alb) showed significant differences (P < 0.05). The sample selection was consistent with the clinical situations of MN patients and normal control subjects.The general clinical characteristics of MN patients and normal control subjects are shown in We first compared the performance of the three arrays in detecting proteins in normal kidney samples.https://doi.org/10.1155/2017/7843584. All 40 proteins could be detected in QAH-INF-3 and the exact signal intensities analysis showed that only 22.5% (9/40) of the signal intensity ratios of 12.5 μg/6.25 μg were between 1.3 and 2.0. Obviously, QAH-INF-9 does not meet our requirements.For QAH-INF-3, the fluorescence image, standard curves, and scatter plot are shown in Supplementary Figure 1 in Supplementary Material available online at μg/25 μg signal intensities was 0.99, which was far less than the theoretical ratio of 2.0. The exact signal intensities ratio of 50 μg/25 μg showed that 26 (5.13%) proteins were not detected, merely 61 (12.03%) were between 1.3 and 3.0, 411 (91.07%) were less than 1.3, and 9 (1.78%) were larger than 3. The majority of the ratios are biased to the lower value.For L-507, the fluorescence image and the signal values are shown as Supplementary Figure 2. The average ratio of the 50 μg/25 μg signal intensities was 2.27, which is close to the theoretical ratio of 2.0. The exact signal intensity ratios of the 50 μg/25 μg results showed that 4 (0.81%) proteins were not detected, 214 (43.41%) were between 1.3 and 3.0  compared to QAH-INF-3  and L-507 , suggesting that L-493 is more suitable for detecting proteins in kidney tissue. Therefore, we selected L-493 to detect DEPs in tissues from MN patients and normal controls.P < 0.05), and they were all upregulated in MN compared to normal tissue (P < 0.05).The fluorescence images of MN and normal control were shown in l tissue . The exal tissue  found seMore importantly, we constructed a molecular network based on protein-protein interactions (PPI) by using STRING . The netP < 0.05), similar to 1.75, 1.91, and 2.06 in L-493, respectively. And immunohistochemistry also showed the overexpression of CTSD and CD46 in MN , as an important component of the intracellular signaling pathway for various immunoreceptors, could also regulate cell adhesion . SerotraIt should be mentioned that our study has some limitations. First, the protein microarray has some deficiencies, of which the most important one is that it cannot distinguish the protein location from glomeruli and tubules in kidney. Second, acquiring samples was really challenging, and high cost of the protein chip limited the sample size in the experiments and validation. In addition, we were also unable to compare multiple disease groups such as MN with IgA nephropathy, minimal change disease (MCD), and focal segmental glomerular sclerosis (FSGS). More studies are needed to address this in the future.In conclusion, we used the protein array to identify differential proteins expressed in kidney tissue from MN patients, screened out proteins that may be related with MN, and reported several possible pathways that could be involved in ECM accumulation. This study is a preliminary exploration, the differential expressed proteins and pathways supply us with more targets to study MN, and we hope to continue pursuing this discovery with more thorough research.The supplementary material provides the proformences of QAH-INF-3 and L-507 with pictures, and the table list of 66 DEPs in MN.


P<0.001) and group B . Lipid index was significantly decreased in both group A  and group B . The incidence of TCFA was decreased statistically in both group A (P <0.001) and group B (P <0.001). The patients with known CHD can equivalently benefit from statins therapy by stabilizing the lipid-rich plaques. Patients with moderate to high FRS might benefit more within the first year from event time.The aim of the study was to investigate the impact of statins therapy on morphological changes of lipid-rich plaques by OCT  in patients with known CHD (coronary heart disease), stratified by FRS. Ninety-seven lipid-rich plaques from sixty-nine patients who received statins therapy and underwent serial OCT images  were divided into 2 groups according to the FRS (framingham risk score): low risk group A , moderate to high risk group B . Fibrous cap thickness (FCT) was measured at its thinnest part 3 times. Baseline characteristics were not different between the 2 groups. FCT sustained increased from baseline to 6-month and 12-month follow up in both group A (59.8±20.4μm, 118.3±62.5μm, and 158.8±83.4μm respectively,  Framingham risk score (FRS), as we known, is a 10-year risk of cardiovascular events which was calculated by sex, age, current smoking status, systolic blood pressure, total cholesterol and high density lipoprotein (HDL) cholesterol . FurtherThe most common lipid-lowering therapy was statins therapy which has been recognized as an effective approach to improve the clinical outcomes of CHD patients by stabilizing vulnerable plaques , 6. IntrWhether known CHD patients with different FRS can benefit equally from statins therapy after event is unknow. Therefore, in this study, we aimed to investigate the impact of statins on morphological changes of lipid-rich plaques by OCT in patients with CHD, stratified by FRS.P=0.001) and higher prevalence of male , hypertension  and smoking  in group B. In addition, all the 3 therapeutic dose is not statistically significant between the 2 groups .A total of 97 lipid rich plaques (LRPs) were detected in 69 patients. There were 45 plaques from 35 patients in group A and 52 plaques from 34 patients in group B. Baseline patient characteristics are shown in Table P=0.010) and the reference vessel diameter (RVD) was also smaller in group B . While the DS% and lesion length was not significantly different between the 2 groups.Baseline angiographic characteristics are showed in Table P<0.001) and group B . Detailedly, there was a significantly decreased from baseline to 6-month in both 2 groups , but from 6-month to 12-month, there was no statistical significance  and group B . In addition, hs-CRP also decreased significantly from baseline to 6 month until 12 month in group A  and group B   and glycosylated hemoglobin (Ha1C) at the 3 time points of the 2 groups in the study are shown in Table e Figure . Similar) Figure . While, P<0.001) and group B . In detail, FCT was significantly sustained growth in both 2 groups from baseline to 6-month  and from 6-month to 12-month  , 6month (P =0.642) and 12 month follow up (P =0.761). In addition, the incidence of thin-cap fibro-atheroma (TCFA) was significantly decreased in both 2 groups under the statin therapy (both P<0.001). For group A, the incidence of TCFA was significantly decreased from baseline to 6-month  but was not different from 6-month to 12-month   and from 6-month to 12-month . When comparing the incidence of TCFA at the 3 time points, it was similar between group A and group B at baseline (P =0.237), 6-month (P =0.798) and 12-month (P =0.955). The incidence of macrophage accumulation and cholesterol crystals decreased significantly from baseline to 12 month in group B  but not in group A , while the change of micro-channel did not reach the statistical significance in both two groups. When comparing the incidence of macrophage accumulation, cholesterol crystals and micro-channel, it was not different between group A and group B at each time point.A total of 97 LRPs (45 in group A and 52 in group B) were included in this study. The OCT characteristics of the LRPs were showed in Table ) Figure . While t) Figure . And it The major findings of the present study were as follows: First, statins therapy could decrease serum levels of atherogenic lipoproteins (LDL-C), TC, TG and inflammation biomarkers (hs-CRP ) from baseline to 6month and 12 month in the 2 groups of CHD patients regardless of risk stratification. Second, statins therapy was associated with a sustained increase in FCT regardless of risk stratification at 6-month and 12-month follow-up and the incidence of TCFA significantly decreased in both 2 groups over time. In addition, lipid index was significantly decreased in both 2 groups. Third, statins therapy could decrease macrophage content, intra-plaque micro-channel and cholesterol crystals. However, changes in macrophage content and cholesterol crystals were only observed in moderate to high risk group patients. The change of intra-plaque micro-channel did not reach the statistical significance in both 2 groups over time.P =0.191). And in low risk group, there were no impact on the change in FCT from baseline to 12-month follow up . However, in moderate to high risk group, there was significant difference in the change of FCT from baseline to 12 month follow up . LRPs in moderate to high risk patients seems more sensitive to intensive statin therapy. However, the change of FCT were comparable between the two groups from baseline to 6 month and 12 month follow up  overall.FRS was a 10-year risk score for development of cardiovascular disease and has been validated in Canada . In thisThe vulnerable plaques has been demonstrated in the pathogenesis of acute coronary syndrome . PlaquesStatins shoud be routinely used regardless of the risk stratification. CHD patients could equivalently benefit from statins therapy by stabilizing of the LRPs regardless of risk stratifications, stratified by 10-year FRS. Moderate to high risk patients would benefit more within the first year after event.There were several limitations in the present study: First, this was a post-hoc sub-analysis of a randomized study, and the study sample is relatively small. Second, only LRPs were enrolled in the present study and potential selection bias is unavoidable. Third, very high-risk patients were excluded from the study, such as those with severe hepatic dysfunction or congestive heart failure. Fourth, due to the practical difficulties of imaging, very distal and ostial segments were not included in our study. Fifth, according to OCT definitions, TCFA was defined as a LRPs with FCT ≤65μm. Macrophages accumulation was defined as a region with signal-rich, distinct or confluent punctuate heterogeneous backward shadows. Due to the limitation of current OCT imaging system, we have to admitted that some fibrous plaques might have been misdiagnosed as TCFA at the presence of macrophage accumulation on the surface of fibrous atherosclerotic plaque. Lastly, the direct relationship between increase in FCT and reduction in coronary events risk remains unknown. Further studies with larger population and longterm follow-up are needed to study the impact of statins on morphological changes of LRPs, stratified by FRS.The present OCT study was a post-hoc sub-analysis of a prospective, open-labeled, randomized trial, which was performed to evaluate the progression of lipid-rich plaques treated by statins at baseline, 6-month, and 12-month follow-up . A totalnd Affiliated Hospital of Harbin Medical University .All the patients were provided with written informed consent and this trial was approved by the Ethics Committee of the 2Coronary angiography was performed by experienced operators from our hospital. All the procedures are radial approach after intracoronary injection of nitroglycerin (100-200 mg). Any non-culprit lesion with 30%-70% diameter stenosis was enrolled in the analysis. Quantitative coronary angiogram analysis (QCA)  software package was used by two angiographers who were blinded to clinical information from an independent core laboratory for coronary angiograms.In the present study, all the OCT procedures were performed in M2/M3 system . A 0.016-inch OCT catheter  was advanced to the distal site of the target lesion through a 3-F occlusion balloon catheter. An occlusion balloon was inflated at 0.4 to 0.6 atm at the proximal site of the plaque was needed to remove red blood cells from the field of view. Lactated Ringer's solution was infused into the coronary artery from the distal tip of the occlusion balloon catheter at 0.5–2.0 mL/s by a high-pressure injector. The vessel was imaged with an automatic pullback device at 3.0 mm/s.An offline software (Light Lab Imaging) was used for OCT image analysis at an independent core laboratory of Massachusetts General Hospital (MGH). OCT images were analyzed at 1 mm interval. All the baseline and follow-up OCT images were analyzed by two independent reviewers who were blinded to clinical information. OCT image was identified according to the criteria of the Clinical Expert Consensus Document on OCT. A third professional investigator intervened when there was any discordance between the observers and a consensus was obtained.The target lesions were determined according to coronary angiography. At 6-month and 12-month follow-up, the corresponding segment for OCT analyzing to baseline imaging were identified on the basis of reliable anatomic marks such as the side branches, calcifications, and stent edges. All the enrolled plaques should be at least 5mm away from the stent edge.Only LRPs was analyzed in this trial. On the OCT image, LRPs was semi-quantified according to the maximal lipid arc. Lipid arc was measured at every 1 mm interval throughout the entire length of each lesion and the values were averaged. All the OCT images were analyzed using the previously validated criteria for plaque characterization. LRPs was defined as the plaque with lipid content > 100° and FCT< 120 μm on OCT image. FCT covering lipid core was measured at its thinnest part 3 times and the average value of the three measurements was used for subsequent analysis. At follow up, FCT was measured at the same site according to the landmark as it was measured at baseline using the same methodology. Lipid length was also measured on longitudinal view. Lipid index (LI) was defined as the averaged lipid arc multiplied by lipid length. The characteristic of lipid core was a diffusely bordered, signal-poor region Figure . TCFA waP-value<0.05 was considered statistically significant.SPSS 19.0  was used for data analysis. Continuous variables were presented as mean ± standard deviation (SD) for normally distributed variables or median (25th-75th percentiles) for non-normally distributed variables. For the normality assessment of continuous variables, the Kolmogorov–Smirnov test was used. Categorical variables were expressed as absolute numbers and percentages. For the association of qualitative variables, chi-square test was used. While for the comparison of continuous results in 2 groups over the three time points, the repeated measures analysis of variance (RM-ANOVA) with the Bonferroni correction for post-hoc comparisons was applied. Take into account that per patient may have multiple plaques, the Generalized Estimating Equations (GEE) was used when comparing the plaque characteristics in 2 groups. A


The immunosuppressive drug tacrolimus has the short-term effect of reducing proteinuria in patients with immunoglobulin A nephropathy (IgAN). Our study investigated the effects on proteinuria and kidney function after discontinuation of tacrolimus.Patients with biopsy-proven IgAN were included in the study and randomly divided into two treatment groups. There was a corresponding control group for each treatment group. The first group included patients treated with tacrolimus (Tac vs non-Tac group) and the second group included patients with a renin angiotensin system blocker (RASi vs non-RASi group). The Tac group received treatment for up to 16 weeks, with the administration of tacrolimus being ceased at the final visit . We tracked the patients at 12, 24, 52, and 240 weeks . The primary outcomes examined were the percentage change  of time-averaged proteinuria  and the estimated glomerular filtration rate (eGFR). Time-averaged proteinuria was defined as the average of urine protein to creatinine ratio (UPCR), measured every 3 months during both the trial and observational phases of the study.p = 0.023 and p = 0.003, respectively). However, the difference between the Tac group and non-Tac group was not evident in the other review periods, estimated by linear mixed effect model. The percentage change in TA-proteinuria was greater in the Tac group than that in the corresponding control group . Therefore, during the observational phase, TA-proteinuria was not significantly different between the Tac group and the non-Tac group . The levels of eGFR throughout the observational phase were not significantly different between the two groups. Furthermore, the mean rate of eGFR change throughout both phases of the study was -6.4 ± 5.9 mL/min/1.73 m2/year in the non-Tac group and -5.4 ± 7.9 mL/min/1.73 m2/year in the Tac group (p = 0.988).A significant reduction in UPCR was observed in the Tac group compared to non-Tac group at the 4 and 8 week visits during the trial phase (The anti-proteinuric effect of tacrolimus was promptly reversed 3 months after discontinuing the drug. The use of tacrolimus for a short period of time for patients with IgAN temporarily reduces proteinuria, but the data showed no long-term efficacy regarding proteinuria reduction and improvement of renal function. Proteinuria is a strong predictor of disease progression and kidney survival in patients with immunoglobulin A nephropathy (IgAN) –3. ReichTacrolimus has previously been administered to patients with kidney glomerular diseases in an attempt to reduce proteinuria. However, the mechanisms of tacrolimus for reducing proteinuria remains uncertain. Two reports from a study carried out in China stated that up-regulation of the core I B3-Gal-T-specific molecular chaperone gene expression can treat IgAN , 7. Wen However, it is not certain whether proteinuria reduction persists after tacrolimus cessation. In this study, we investigated the changes in proteinuria and kidney function after discontinuation of tacrolimus.2, urine albumin to creatinine ratio (UACR) ≥ 0.3 and < 3.0 g/g cr, and blood pressure (BP) less than 130/80 mmHg during the 3-month period before randomization. The eGFR was estimated using the 2009 CKD-EPI cr equation and the IDMS-traceable cr measurement [The data used in this study were from a previous double-blind randomized controlled clinical trial to verify the anti-proteinuric effect of tacrolimus in patients with IgAN . The study protocol was described in a previous report . Brieflysurement . The iniThe study protocol complied with the Declaration of Helsinki and received full approval from the Seoul National University Hospital’s institutional review board (IRB number: H-1002-032-309), which waived the need for informed consent since the study did not infringe on patient privacy or health status.All patients were periodically assessed within our hospital. We gathered all clinical parameters regarding medication, urine protein to creatinine ratio (UPCR), UACR, and serum cr from the hospital database of electronic medical records . During 2/year detected during the observational phase. The remission of proteinuria was defined as a UPCR reading of < 0.2 g/g cr at least twice with ≥ 1 month interval. The composite outcome of renal dysfunction was defined as an increase in serum cr level noted during the observational phase whereby there was a ≥ 50% change from baseline  or deterioration of renal function to end stage renal disease.Time-averaged proteinuria was defined as the average of UPCR, measured every 3 months during both the trial and observational phases of the study. Furthermore, we calculated the percentage change in TA-proteinuria between the trial and observational phases of the study. We calculated the mean rate of eGFR change at each follow-up during the observational phase as compared to the initial level of eGFR at trial phase. A rapid decline in eGFR was defined as a change of ≥ 5 mL/min/1.73 mAll statistical analyses were performed using SPSS version 22 . We used the Mann-Whitney test for analysis of continuous variables and Pearson’s Chi-square test or Fisher’s exact test for qualitative variables. The data throughout the follow-up period was analyzed with linear mixed effect model. To define independent factors related to rapid decline of eGFR, we used multiple logistic regression analysis adjusted with traditional risk factors such as blood pressures, eGFR and UPCR at the initial examination of the trial phase, and TA-proteinuria during the observational phase, and follow-up duration. We used the Cox's proportional hazard model to define independent risk factors for the composite outcome of renal dysfunction adjusted with traditional risk factors. A p-value < 0.05 was used as a criterion of significant difference.We enrolled 37 out of 40 patients who had completed the trial to evaluate the anti-proteinuric effect of tacrolimus in patients with IgAN . The chaTac: Tacrolimus, RASi: renin-angiotensin-aldosterone system inhibitor, SBP: systolic blood pressure, DB: diastolic blood pressure, cr: creatinine, UPCR: urine protein to creatinine ratio, TA-proteinuria: time averaged proteinuria calculated as the mean urine protein to creatinine ratio, measured every 3 months during both the trial phase of the study, UACR: urine albumin to creatinine ratio, eGFR: estimated glomerular filtration rate by the equation of CKD-EPI, CRP: C-reactive protein, Hematuria: defined as RBC ≥5 under 400-fold magnification examined by light microscopy. P-values were estimated using the Mann-Whitney test.The patients were followed up for an average of 57.9 ± 13.8 months (range: 22.8–66.7), until October 2016. Among the patients in the Tac group, tacrolimus was stopped at the last visit of the trial phase in 13/18 patients, within 15 days after the last visit in 4/18 patients, and at 15.8 months after the last visit in 1/18 patients who wanted to use it for a longer period. During the observational phase, serum uric acid lowering medication was prescribed more frequently in the Tac group compared to the non-Tac group  . Anti-hy2/year in the non-Tac group and -5.4 ± 7.9 mL/min/1.73 m2/year in the Tac group (p = 0.988).The UPCR was lower in the Tac group compared to the corresponding control group at the 4-week and 8-week visits during the trial phase . However, this difference between the Tac and non-Tac group was not evident at the other follow-up periods, estimated by linear mixed effect model . The UPC2/year in the non-RASi group and -3.0 mL/min/1.73 m2/year in the RASi group (p = 0.022).The UPCRs were not significantly different between the RASi group and the corresponding control group throughout both the trial and observational phases . During We compared four outcomes between the groups . The nonIn our previous study, tacrolimus had an anti-proteinuric effect during 16 weeks of treatment  of patients with IgAN patients and mild to moderate proteinuria . After t2. In these patients, podocyte injury may be mild, and therefore the effect of tacrolimus induced kidney protection may not be obvious.Tacrolimus was effective in controlling proteinuria in patients with IgAN –13. ReceIn this study, the 3-month treatment did not cause a difference in TA-proteinuria and eGFR between the Tac group and the non-Tac group. Based on several other studies demonstrating the efficacy of tacrolimus, the trial phase would not be a long enough in duration to result in long term effects. However, at present, no guidelines have been established and thus treatment period varies from 3 months to 1 year , 13, 26.Another study revealed that after cessation of a 6-month tacrolimus treatment, proteinuria relapsed in 4/9 (44.4%) patients . The timRASi was the main drug for controlling blood pressure and proteinuria in patients with IgAN . There wThis study was limited by the patient population, which had biopsy confirmed IgAN with mild to moderate proteinuria. Hence, our results not necessarily be generalized to all patients with IgAN. Furthermore, we were not able to confirm pathologic changes through biopsy, after the trial and observational phases. Identifying pathologic changes through additional animal experiments using different drug dose and treatment duration, may be the basis for our results that the tacrolimus will not show a prolong effect.In conclusion, the anti-proteinuric effect of tacrolimus was promptly reversed 3 months after discontinuation of the drug. The use of tacrolimus for a short period of time for patients with IgAN and mild to moderate proteinuria, temporarily reduces proteinuria, but there seems to be no long-term efficacy.S1 File(XLSX)Click here for additional data file.


Background. Electroacupuncture (EA) at ST36 has been used to regulate gastric motility and effectively improve gastric emptying in diabetic patients. Nevertheless, the specific mechanisms underlying the efficacy of this treatment remain unknown. The aim of this study was to assess the variations of interstitial cells of Cajal (ICC) and explore the changes in mSCF/KIT-ETV1 signaling in the antrum and corpus of diabetic mice after treatment with EA. Methods. Male C57BL/6 mice were randomized into five groups: control group, diabetic group (DM), diabetic-plus-sham EA group (SEA), diabetic-plus-low-frequency EA group (LEA), and diabetic-plus-high-frequency EA group (HEA). The expression levels of Ano1, c-Kit, and ETV1 were assessed by immunofluorescence in the antrum and corpus. Western blotting and PCR methods were further used to evaluate c-Kit, mSCF, and ETV1 expression. Results. (1) c-Kit and Ano1 were obviously decreased in the DM group, but c-Kit reduced much more than Ano1. (2) The mSCF, c-Kit, and ETV1 mRNA and protein levels were obviously decreased in the DM group in both the antrum and the corpus (P < 0.01), but they were significantly elevated in the LEA and HEA groups (P < 0.01). Conclusions. Ano1 is a reliable marker to detect ICC changes in diabetes; low- and high-frequency EA at acupoint ST36 can protect the networks of ICC possibly via normal activation of mSCF/KIT-ETV1 signaling. The incidence rate of diabetes has been rapidly increasing following the improvements in the standard of living in recent decades. The gastrointestinal (GI) complications of diabetes are primary causes of the lower quality of life in diabetic patients, as well as increased hospitalization and mortality rates. Gastroparesis, defined as delayed gastric emptying without mechanical obstruction, is the most common complication in the stomach due to long-term chronic hyperglycaemia. A series of studies about the mechanisms underlying gastroparesis have been proposed, and different types of injured cells in the stomach may contribute to gastroparesis , 2.Injury of the interstitial cells of Cajal may be one of the most significant factors causing gastrointestinal dysfunction in diabetic patients. Normal gastric emptying depends on the coordination of the contractile activity of the corpus and antrum. Specifically destroying ICC in the antrum and corpus has revealed slow wave dysrhythmia and delayed gastric emptying , 4. Gast−/− mice have been shown to have losses of the intramuscular ICC and myenteric ICC [A few studies have demonstrated that SCF/Kit signaling was necessary for sustaining the ICC phenotypes, proliferation, and differentiation . ICC loseric ICC . So far,Although pharmaceutical intervention and some nondrug treatments have been utilized, the curative effects of diabetic gastroparesis are still unsatisfactory. Electroacupuncture (EA) is currently used as an optimized type of acupuncture and has revealed a potential role in regulating gastric motility and treating gastroparesis , 11. In The purposes of this study were to (1) employ c-Kit combined Ano1 to evaluate the changes of ICC in diabetic mice; (2) investigate whether the mSCF/Kit-ETV1 signaling is involved in the protection of the ICC networks in the antrum and corpus of diabetic mice after electroacupuncture at ST36.A total of 60 male six- to eight-week-old C57BL/6 mice were used in this study. All of the mice were obtained from Beijing Hua Fu Kang Bio-technology Co., LTD, Beijing, China. They were housed in a standardized laboratory room  and were given food and sterile water ad libitum. Before entering the formal study, the mice had adapted to the laboratory environment for two weeks. The mice received humane care and all of the experiments were approved by the Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology.After overnight fasting without water-deprivation, diabetes was successfully induced in mice by a single intraperitoneal injection of streptozotocin  at a dose of 150 mg/kg that was prepared in 0.1 mol/L citrate buffer . The control group was injected with the same volume of citrate buffer. One week after the injection, diabetes was confirmed by measuring the blood glucose level using a drop of whole blood obtained from a small skin incision made at the tip of the tail. Animals were considered diabetic if their blood glucose level was above 250 mg/dL.All of the mice were randomly divided into five groups (12 mice/group): the control group, the diabetic group (DM), a group of diabetic mice with sham EA , a group of diabetic mice treated with low-frequency EA group , and a group of diabetic mice treated with high-frequency EA group . EA was carried out continuously at ST36 for eight weeks. Eight weeks later, all of the mice were sacrificed and specimens of the antrum and corpus were carefully obtained. Part of fresh tissues were used for immunofluorescence. Other part of tissues were stored at −80°C for western blotting and RT-PCR analysis.Electroacupuncture was performed at 9:00–9:30 AM every day. An electrical stimulator  was used to stimulate. The acupoint of ST36 for the mice was located at the posterolateral knee of bilateral hind limbs, approximately 2 mm below the fibular head . For the3, 1.0 NaH2PO4, 1.2 MgSO4, 11.1 Glucose, 2.5 CaCl2, and pH 7.3–7.4 after being bubbled with 95% O2 and 5% CO2. The stomach was opened along the lesser curvature and the gastric contents were washed away. The tissue was then pinned in a dish that was coated with Sylgard with the mucosa face up. The mucosa was carefully peeled away with forceps. The antrum and corpus whole-mount preparations were fixed with ice-cold acetone for ten minutes. Following fixation, the preparations were rinsed three times (ten minutes each time) in 1x PBS. Nonspecific binding was blocked in PBS containing 5% normal goat serum and 0.5% Triton X-100. The tissues were next incubated for 48 hours at 4°C with the primary antibody diluted in primary antibody dilution buffer containing 0.3% Triton X-100. There were two pairs of primary antibodies used: rat anti-c-Kit  with rabbit anti-Ano1 , rat anti-c-Kit , and rabbit anti-ETV1  that were used for incubations. After washing with PBS, the specific labelling was evaluated by incubation with Dylight 488 with goat anti-rat IgG (Abbkine) and Dylight 594 with goat anti-rabbit IgG (Abbkine) diluted in PBS containing 0.5% Triton X-100 for two hours. The incubation solution without primary antibodies was regarded as the negative control. A confocal microscope  was used to examine the preparations.Tissues of four or five mice for each group were used to perform immunofluorescence staining. The freshly obtained mouse stomachs were placed in Kreb's solution (mmol/L): 118.1 NaCl, 4.8 KCl, 25 NaHCOTissues of five mice for each group were applied to western blots analysis. Fresh-frozen antrum and corpus specimens were homogenized separately in RIPA buffer with protease inhibitor. Then, the homogenates were centrifuged at 12000 rpm for 15 min at 4°C and all of the supernatants were collected as the protein. The concentration of the protein was measured by the bicinchoninic acid (BCA) method. Next, equivalents of 80 mg of extracted proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred to PVDF membranes. The nonspecific binding sites were subsequently blocked in 5% nonfat dry milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (TBST). One hour later, all of the membranes were incubated with primary antibodies polyclonal goat anti-c-Kit , goat anti-SCF , or rabbit anti-ETV1  overnight at 4°C. Rabbit anti-mouse GAPDH  served as the internal control. After being washed three times in TBST, the membranes were incubated with a HRP-linked secondary antibody  for one hour at room temperature. After being washed three times in TBST, detection of the bands was performed by a chemical reaction with the enhanced chemiluminescence reagents , and the blot was subjected to autoradiography. The Quantity One software  was applied to measure the band intensity.R480 . The specific primer sequences used were as follows: c-Kit mRNA-5′-GACCCGACGCAACTTCCTTA-3′ and 5′-GAGCATCTTCACGGCAACTGT-3′, mSCF mRNA-5′-GGAAAATAGTGGATGACCTCGTG-3′ and 5′-TGGAATCTTTCTCGGGACCTAAT-3′, ETV1 mRNA-5′-GTCCATACCAGACAGCACCTACC-3′ and 5′-GAA GGGGATGTTTGGCTCAG-3′, GAPDH mRNA-5′-TTCACCACCATGGAGAAGGC-3′, and 5′-GGCATGGACTGTGGTCATGA-3′. The transcript level was normalized by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the relative expression level of each target gene was calculated with the power formula: 2−ΔCT (ΔCT = CTTarget−CTGAPDH).Tissues of six mice were used to implement quantitative real-time PCR analysis. The total RNA of the antrum and corpus tissues from mice were extracted with the TRIzol reagent . Then, the RNA was converted into cDNA using PrimeScript RT Master Mix . Real-time quantitative reverse-transcriptase PCR (qRT-PCR) was performed using SYBR Premix Ex TaqII (Takara) on a Roche LightCyclerP < 0.05 was regarded to be statistically significant. The statistical analysis was performed using with SPSS 17.0 .All values were expressed as the means ± SEM and a one-way ANOVA was applied to compare the differences among multiple groups. Pearson's correlation and linear regressions were used to evaluate the relationships between the mSCF and ETV1 protein expression. A value of Double labelling of c-Kit and Ano1 Figures  was perfP < 0.01) and the corpus (P < 0.01)  and corpus (both P < 0.01). However, there were no significant differences in the protein expression of c-Kit between the SEA group and the DM group in either the antrum (P = 0.718) or the corpus (P = 0.797). The mRNA expression showed a tendency similar to the protein expression in the antrum and corpus  and corpus (P < 0.01) in the DM group. However, compared to the DM group, the expression of mSCF protein was dramatically elevated in both the LEA and the HEA groups in both the antrum (both P < 0.01) and corpus . There were also no significant differences between the SEA group and the DM group in terms of the expression level of mSCF protein in either the antrum (P = 0.603) or corpus (P = 0.478). An analogous situation was found for the mRNA expression  (P = 0.000)  and the corpus (both P < 0.01). However, no differences were found in the ETV1 expression levels between the DM group and the SEA group in either the antrum (P = 0.182) or the corpus (P = 0.624). Similar changes in ETV1 mRNA expression were observed in the antrum and corpus   and corp= 0.000)  compared Figures .r = 0.917 (P = 0.000) and r = 0.940 (P = 0.000), resp.). In the linear regression analysis, mSCF was entered as a dependent variable for both the antrum     and corp< 0.001) . The regIn our current study, we have accurately exhibited the changes of the networks of the ICC and the expression of molecules involved in mSCF/Kit-ETV1 signaling in the antrum and corpus of STZ-induced diabetic mice. After the mice were treated with EA, the mSCF/Kit-ETV1 signaling sustained the normal activated state and networks of the ICC to near the normal level.Acupuncture is a traditional Chinese medicine that has been used for thousands of years. Electroacupuncture, which is a modified form of acupuncture, has recently attracted many researchers' attention due to its safety and cost-effectiveness. On the aspect of regulating gastrointestinal motility, ST36 (Zusanli) is the most commonly used point and different frequencies are employed. For example, several studies have demonstrated that gastric emptying could be accelerated by low-frequency EA at ST36 in animal models , 15. ButV mice, a few Ano1 positive c-Kit dim or negative cells were found [Antibodies against c-Kit have been extensively applied to characterize the ICC changes in diabetes. Recently, a few studies have adopted Ano1 to detect all classes of ICC in the stomachs of humans and mice . Ano1 isre found , 21. Thure found . Perfectre found , 23. ThuThe stem cell factor (SCF), as the ligand of c-Kit, can activate the c-Kit receptor protein and sustain ICC development. ICC proliferation and differentiation have been proposed to result from the activation of SCF/Kit signaling . Two forAs mentioned above, ETV1 is the major downstream effector of mSCF/Kit signaling. Nevertheless, at present, no reports about ETV1 have been published in models of diabetes and EA. This is because, as an ETS family transcription factor, the focus on ETV1 has been on ETS-dependent tumours, especially for the development of gastrointestinal stromal tumours , 26. In In conclusion, the decreased ICC networks in the antrum and corpus of diabetic mice could be well detected using c-Kit staining, and Ano1 is also a reliable and precise marker to monitor the changes of the ICC in diabetic mice. The increased ICC in the low- and high-frequency groups stained simultaneously by Ano1 and c-Kit further enhanced the effects of EA. The normal activation of mSCF/Kit-ETV1 signaling by low- and high-frequency EA may protect the networks of the ICC in diabetic mice at least in part. That may be a new view for us to understand the underlying mechanisms of EA and a deep investigation needs to be carried out in the future.


Klebsiella pneumoniae is a common cause of nosocomial infections worldwide. Recent years have seen an explosion of resistance to extended-spectrum β-lactamases (ESBLs) and emergence of carbapenem resistance. Here, we examine 198 invasive K. pneumoniae isolates collected from over a decade in Kilifi County Hospital (KCH) in Kenya. We observe a significant increase in MDR K. pneumoniae isolates, particularly to third generation cephalosporins conferred by ESBLs. Using whole-genome sequences, we describe the population structure and the distribution of antimicrobial resistance genes within it. More than half of the isolates examined in this study were ESBL-positive, encoding CTX-M-15, SHV-2, SHV-12 and SHV-27, and 79% were MDR conferring resistance to at least three antimicrobial classes. Although no isolates in our dataset were found to be resistant to carbapenems we did find a plasmid with the genetic architecture of a known New Delhi metallo-β-lactamase-1 (NDM)-carrying plasmid in 25 isolates. In the absence of carbapenem use in KCH and because of the instability of the NDM-1 gene in the plasmid, the NDM-1 gene has been lost in these isolates. Our data suggests that isolates that encode NDM-1 could be present in the population; should carbapenems be introduced as treatment in public hospitals in Kenya, resistance is likely to ensue rapidly.Multidrug resistant (MDR)  Enterob problem . In KCH idelines  of penicK. pneumoniae is emerging across the globe  found in 22.1The study was conducted at Kilifi County Hospital (KCH) in rural Kilifi, one of Kenya’s poorest counties, where a majority of the population are rural farmers. Kilifi is 60 km from Mombasa, the nearest city with a secondary government hospital and several private secondary hospitals. KCH is a primary government hospital that serves a population of about one million. The KEMRI-Wellcome Trust Research Programme (KWTRP) has carried out surveillance of invasive bacterial infections at KCH for paediatrics since 1994 and for adults since 2007 .At KCH, blood samples are routinely collected for culture for consenting paediatric and adult medical patients at admission . Cerebrospinal fluid and other sterile site samples are collected and cultured on admission if clinically indicated and, likewise, throughout the course of the illness subsequent specimens are collected for culture at the discretion of the attending clinician.For the purpose of this analysis duplicate isolates from the same patient were not included. Blood cultures were performed using the automated BACTEC system  and isolates were identified using standard microbiological techniques . Community-acquired infection was defined as culture-positive samples collected upon admission or less than 48 h after admission. Hospital-acquired infection was defined as culture-positive samples collected from patients who had been admitted to the hospital at least 48 h before sample collection, or had been discharged from hospital within two weeks of the present admission or had been born in KCH and admitted directly to the neonatal unit.2.2K. pneumoniae is intrinsically resistant to ampicillin.Antimicrobial susceptibility was determined by a broth microdilution minimum inhibitory concentration (MIC) method  in accordance with the Clinical Laboratory Standards Institute (CLSI) guidelines . Extende2.3K. pneumoniae isolates grown on Cysteine Lactose Electrolyte-Deficient (CLED) agar were harvested and emulsified in 500 μL 1% Tris EDTA to form a 0.5 McFarland solution. Samples were centrifuged at 5340 Rcf for 10 min. The supernatants were discarded while the pellets were extracted using the QIAamp® DNA mini kit  according to the manufacturer’s instructions for isolation of genomic DNA from bacterial cultures. DNA was eluted in 60 μL and quantified using an Invitrogen Qubit® 2.0 Fluorometer . The DNA for 190 isolates was subjected to paired-end whole genome sequencing on an Illumina HiSeq2000 Platform at the Wellcome Trust Sanger Institute  to generate 100 bp paired-end reads. DNA for eight isolates were sequenced on Illumina MiSeq to generate 150 bp paired-end reads (Overnight cultures of nd reads .2.4K. pneumoniae MGH 78578 (RefSeq: GCF_000016305.1) genome using a pipeline developed in-house. Reads were aligned using SMALT . The isolates were assembled using an in-house pipeline that included Velvet v1.2.09 . Clonal complexes (CC), defined as a group of profiles that differ by no more than one gene from at least one other profile of the group, were determined from the MLST profiles of isolates with known alleles. The CC nomenclature used is specific to this study.For Multi-Locus Sequence Typing (MLST) all genomes were mapped against the seven housekeeping genes, T scheme . The seq2.7K. pneumoniae using RAxML v7.8.6  was used to search de novo assembled contigs against the non-redundant resistome, which comprised of 1777 resistance genes. Search hits with >90% nucleotide identity over >80% of the resistance gene, were considered a match.The resfinder database of bacterial antimicrobial genes  was manu2.9JN420336.1) and that of pNDM-KN (GenBank accession: JN157804) using mapping methods described above. Contigs from the de novo assemblies of isolates that mapped with read coverage of ≥6 reads over 70% or more of the plasmid sequence were searched against the plasmid sequence using MUMMER v3.1. A contig was considered to be well mapped to the plasmid if at least 80% of the contig length aligned to the plasmid with a minimum of 90% nucleotide identity. We considered an isolate with coverage of at least 60% to the plasmid to strongly suggest the presence of the plasmid.Reads from all isolates were mapped against the pNDM-MAR sequence (GenBank accession: 33.1K. pneumoniae was isolated from cultures of blood or CSF from 197 (0.4%) children and 24 (0.4%) adults and stored at −80 C. One hundred and seventy four (88%) isolates from children and all the isolates from adults were successfully located and subcultured and subsequently sequenced. The sample set consisted of near-equal proportions of hospital-acquired infections  and community-acquired infections .A total of 52,610 paediatric (0–14 years) and 16,076 adult patients were admitted to KCH in the study periods of 2001–2011 and 2007–2011, respectively. Of these, 92%  paediatric and 54% (8676) of adult patients were eligible for blood culture of which 97%  paediatric and 64% (5594) of adult patients consented to blood tests. Blood samples were collected from 97%  paediatric and 94% (5239) of adult consenting patients. Of these, K. pneumoniae isolates per year by acquisition status and the proportion of isolates that were phenotypically ESBL-positive. These data show that not only did the proportion of hospital-acquired K. pneumoniae increase between 2001 and 2011 but there was an overall increase in the number of ESBL-carrying K. pneumoniae isolates. For both community-acquired infections (p = 0.003) and hospital-acquired infections (p < 0.001), the proportion of ESBL-positive isolates increased from 18% (2/11) and 67% (2/3), respectively, in 2001 to 44% (4/9) and 94% (29/31) in 2011. Although no isolates were found to be phenotypically resistant to carbapenems, 79% (N = 159) were multi-drug resistant (MDR), conferring resistance to between three and 11 agents (excluding penicillins) of the 15 antimicrobial categories tested. MDR was observed in 94% (95/101) of isolates of hospital-acquired infections and 63% (61/97) of isolates of community-acquired infections (p < 0.0001).The antibiotic susceptibility profile for all isolates is summarised in 3.2K. pneumoniae isolates were obtained. Three isolates were excluded from further analyses due to having genome assemblies with greater than 2000 contigs and an average contig length of <3000 bp. The assemblies of the remaining 195 isolates had a median of 85 contigs (range: 22–2895) and N50 of 282,488 . By MLST analysis, 58 distinct STs were represented in 130 isolates; 34 STs in 71 isolates from hospital-acquired infections and 36 STs in 59 isolates from community-acquired infections. Novel STs were found in 28 isolates and 52 novel alleles were found in 37 isolates. Therefore, a total of 138 distinct STs were distinguished. The isolates grouped into 12 clonal complexes (CC), comprising a maximum of three STs and 88 singletons (Table S1). A majority of the CC had approximately equal proportions of community and hospital-acquired isolates, however three were comprised of only community-acquired isolates and one of solely hospital-acquired isolates. STs of half the isolates occurred only once in the decade. However, STs such as ST15 (CC3) and ST17 (CC5) were seen to recur almost every year since their emergence , 29 (9.1%), 70 (14.7%), 61 (14.1%), 43 (10.2%), 52 (10.4%), 93 (24%) polymorphic sites, respectively. Three insertion/deletion (indels) events at the tonB locus  were observed. Excluding these indels 372 of 3012 bp (12.4%) were polymorphic when the seven loci were concatenated. In isolates from hospital-acquired infections and community-acquired infections 335  and 326  sites were polymorphic, respectively. This high genetic diversity observed in MLST groupings was in complete agreement with clustering defined by core genes obtained from whole genome sequencing. The core gene set of 1874 genes was derived from a comparison of all of the coding sequences found in the whole genomes of the 195 isolates. Within 1.2 Mb comprising the core gene set 149,545 (14%) nucleotide sites varied. In addition, 17,439 unique genes were identified among the 195 isolates.Together with the large number of distinct STs in the sample set, considerable sequence diversity was also observed. Loci gyrA and parC genes has identified three phylogenetic lineages of K. pneumoniae globally to date  ESBL-positive strain affected four infants within a span of one month in the neonatal unit, including two fatalities. Isolates from three of the patients were from hospital-acquired infections by formal definition, while the fourth patient was born in the hospital prior to being admitted to the ward. The core genome of all four isolates was identical . A seconA series of hospital-acquired infections caused by an MDR isolate belonging to ST70 (singleton) affected six neonates within 15 days in 2011. This ST was also seen on one previous occasion in a hospital-acquired infection in 2009, sharing the same phenotypic and genotypic antimicrobial resistance profiles, and having a near-identical core genome content (99.99% nucleotide identity) to the 2011 isolates.3.4K. pneumoniae isolates taken from KCH  profile, that of a “wild-type” K. pneumoniae.Phenotypic antimicrobial testing revealed that there were 8 classes of antimicrobial resistance genes present in the from KCH . Genes wK. pneumoniae lineages KPI, KPII and KPIII has been shown to correspond to that of its innate chromosomal β-lactamase genes blaSHV, blaOKP and blaLEN, respectively . Most of these belonged to the 2b group of the Bush and Jacoby functional classification scheme . In all the five isolates the entire ∼14 kb locus, from the chromosomal ygb genes to the lac operon were present and their order maintained. In addition, the latter region was assembled into one contig in all five isolates. Furthermore, copies of intact or truncated transposons were not found in any of the contigs in which this locus was assembled. The above points strongly support the chromosomal origin of the contigs. In three of these isolates multiple copies of the blaSHV gene, accompanied by flanking genes, some truncated, were located at the same locus. A full-length copy of blaSHV genes belonging to the 2be group was also present at the same locus. In the remaining three isolates from the KPI lineage a 2be blaSHV gene had replaced the 2b variant. The blaSHV group 2be genes to date have largely been described as plasmid-encoded genes; they have not been identified as chromosomally encoded in K. pneumoniae.The phylogeny of ectively . These cn scheme  denotingblaOKP β-lactamase gene, blaOKP-A or blaOKP-B, they carried . The mean pairwise nucleotide divergence within the sequences in KPIII-a and KPIII-b was 0.5% and 2.6%, respectively. Even though they shared the same class of chromosomal β-lactamase gene as KPIII-a, isolates in KPIII-b were genetically closer to KPI (mean pairwise nucleotide divergence = 4.4%) than they were to KPIII-a (mean pairwise nucleotide divergence = 4.6%) or to KPII (mean pairwise nucleotide divergence = 5.2%) suggesting that KPIII-b might share a nearest common ancestor with KPI rather than with KPIII-a. Phylogeny of the blaLEN genes of the nine KPIII isolates correlated with that of the core genes .The mean pairwise nucleotide divergence as estimated using the core genes was 0.4% and 1.4% within KPI and KPII lineages, respectively. Within the KPII lineage, Kilifi isolates separated into two known subgroups distinguished by the variant of chromosomal  carried . The ninblaSHV, seven classes of β-lactamase genes often carried on a plasmid were also found in these isolates. These were blaCTX-M, OXAbla, blaTEM, blaDHA1, blaCARB, blaCMY and SblaCO. Of the clinically significant β-lactamases blaTEM-1, an enzyme that can hydrolyze benzylpenicillin and early cephalosporins, was present in 134 isolates (68%). Only one variant of the blaTEM-1 gene was found. A blaCTX-M-15 ESBL encoding gene was present in 92 (47%) isolates and all but one of the isolates carrying a blaCTX-M-15 gene had an ESBL phenotype. Four types of blaOXA genes were found in 61 isolates. Two blaOXA variants, OXA-1 and OXA-10, belong to the 2d phenotypic group (increased hydrolysis of oxacillin), while two variants, OXA-89 and OXA-58 belong to the 2df group that has oxacillinase as well as some weak carbapenemase activity.In addition to 3.5ndm-1. Consistent with this the IncHIB-like and IncFIB- like plasmid replication genes, characteristic of pNDM-MAR, were present in all 25 of these isolates and absent from all other isolates sequenced here. It is clear from ndm-1 cassette from genes ndm-1 to yhxD, and includes the mercury resistance operon and several transposase-encoding regions. AMR genes catB, blaOXA-1 and aac(6′)-Ib are located in tandem on pNDM-MAR, supposedly co-mobilized by IS26. In ten isolates these AMR genes were located on the same contig and they were flanked by inverted repeats of IS26. The blaCTX-M-15 gene in pNDM-MAR is flanked by mobile genetic elements (MGEs) ISEcp1 and Tn3. Nine out of ten isolates carrying the catB-blaOXA-1-aac(6′)-Ib cassette also carried blaCTX-M-15. In all nine isolates blaCTX-M-15 was flanked by a Tn3 transposable element. Although the plasmid appeared to be present, none of the isolates carried the ndm-1 gene itself. The plasmid was detected in multiple STs including ST15 (CC3), ST54 (singleton), and 15 other known and 6 novel ST’s. These included isolates from lineages KPI, KPII and KPIII (data not shown).Of the 195 isolates sequenced, 25 isolates had reads that mapped to between 72%–88% of the length of the pNDM-MAR plasmid known to carry the metallo β-lactamase gene 4K. pneumoniae bloodstream infection at Kilifi County Hospital over a decade, with a concomitant increase in antibiotic resistance. This increase occurred in parallel with an increase in maternity services so that more babies were being born at KCH and unwell neonates were increasingly referred to the paediatric ward for investigation with blood cultures. By 2011, 94% of hospital-acquired and 44% of community-acquired K. pneumoniae bloodstream infections were ESBL-positive, resistant to both first-line and second-line empiric antibiotic treatment. Outbreaks of ESBL-carrying hospital-acquired K. pneumoniae infections, suspected clinically, have been confirmed genomically. We have demonstrated high genetic diversity of K. pneumoniae invasive isolates in Kilifi, mirroring the high global diversity of this successful pathogen.In Kilifi, as in the rest of the world, multidrug resistant Gram-negative enteric bacteria are of mounting concern. We found dramatically increasing numbers of K. pneumoniae were getting a foothold in the region; largely undetected due to a lack of microbiology laboratory capacity. The resulting selection pressure may have increased the generation and spread of ESBL-positive pathogens resistant to ceftriaxone. Our data suggest that multidrug resistance, to ampicillin, gentamicin, tetracycline, trimethoprim-sulfamethoxazole and ceftriaxone, is common among bloodstream isolates of K. pneumoniae in Kilifi. Isolates remained relatively susceptible to ciprofloxacin and amikacin; while not ideal treatment and not without potential problems in children, this was fortunate since carbapenems are largely not available at KCH and do not feature on the WHO essential medicines list or Kenyan National Formulary.Well-equipped microbiology facilities are not commonly found at public hospitals throughout sub-Saharan Africa, so there is a paucity of data on the spread of multidrug resistant Gram-negative pathogens in the region, despite increasing global anxiety. Previous reports of widespread resistance to common first line agents such as ampicillin, trimethoprim-sulfamethoxazole and gentamicin led some hospitals to introduce third generation cephalosporins such as ceftriaxone as second or even first-line empiric antibiotic regimens for suspected infection. We speculate that this approach was probably taking place at the same time as ESBL-carrying Gram-negative bacteria like K. pneumoniae invasive infections in Kilifi cannot be attributed to any one particular ST for either community or hospital-acquired infections. The ubiquitous nature of this pathogen has also been reported in other studies  is a broad spectrum β-lactamase able to hydrolyse all β-lactams, . The increasingly limited treatment options for MDR K. pneumoniae and the availability of off-patent imipenem, may subsequently result in the introduction of carbapenems at KCH. When this occurs the introduction of such a plasmid, bearing NDM, may quickly trigger the selection and spread of carbapenem resistance  under the study accession numbers detailed in Table S1.WGS data is available at the European Nucleotide Archive (ENA) (


Modifications of virus-like nanoparticles (VLNPs) using chemical conjugation techniques have brought the field of virology closer to nanotechnology. The huge surface area to volume ratio of VLNPs permits multiple copies of a targeting ligand and drugs to be attached per nanoparticle. By exploring the chemistry of truncated hepatitis B core antigen (tHBcAg) VLNPs, doxorubicin (DOX) was coupled covalently to the external surface of these nanoparticles via carboxylate groups. About 1600 DOX molecules were conjugated on each tHBcAg VLNP. Then, folic acid (FA) was conjugated to lysine residues of tHBcAg VLNPs to target the nanoparticles to cancer cells over-expressing folic acid receptor (FR). The result demonstrated that the dual bioconjugated tHBcAg VLNPs increased the accumulation and uptake of DOX in the human cervical and colorectal cancer cell lines compared with free DOX, resulting in enhanced cytotoxicity of DOX towards these cells. The fabrication of these dual bioconjugated nanoparticles is simple, and drugs can be easily conjugated with a high coupling efficacy to the VLNPs without any limitation with respect to the cargo’s size or charge, as compared with the pH-responsive system based on tHBcAg VLNPs. These dual bioconjugated nanoparticles also have the potential to be modified for other combinatorial drug deliveries. FA-tHBcAg-DOX VLNPs showed a higher toxicity against both cancer HeLa and HT29 cells. The IC 0.10 μM a. The ICectively b. The ICectively c. The ICectively d.In this study, a dual conjugated drug delivery system was developed by taking advantage of the reactive surface groups of tHBcAg VLNPs a. ApproxLive cell imaging revealed that the cellular uptake of dual conjugated FA-tHBcAg-DOX VLNPs by the cancer cells (HeLa and HT29) was much higher compared with free DOX. This indicates that the uptake of these nanoparticles by the cancer cells was via FR-mediated endocytosis. On the other hand, the cellular uptake of these dual conjugated VLNPs by the normal cells (3T3 and CCD-112) was much lower compared with the cancer cells. This can be explained by the fact that the FR is highly expressed in HeLa and HT29 cancer cells , but it 50DOX of FA-tHBcAg-DOX VLNPs was about two-fold lower compared with that of free DOX. This decrement could be explained by the uptake mechanism of FA-tHBcAg-DOX VLNPs and free DOX. Free DOX molecules diffuse through HeLa and HT29 cell membranes and target the nucleus, while FA-tHBcAg-DOX VLNPs can enter the cells via FR-mediated endocytosis by binding to FR which is highly expressed in HeLa and HT29 cells. Therefore, FA-tHBcAg-DOX VLNPs enhance the cellular uptake of DOX and increase its efficiency at low drug concentrations. By contrast, both the normal 3T3 and CCD-112 cells incubated with FA-tHBcAg-DOX VLNPs showed a conferred protection against DOX, as the IC50DOX of this formulation increased by approximately 2.20 and 2.47-fold, respectively, compared with that of free DOX. Bredehorst et al. [The dual conjugated FA-tHBcAg-DOX VLNPs were more cytotoxic to cancer HeLa and HT29 cells compared with free DOX. The ICt et al.  demonstrt et al.  reportedt et al. . These mIn a previous study, we established a pH-responsive drug delivery system. FA was conjugated to a pentadecapeptide containing nanoglue bound to tHBcAg VLNPs, and DOX was loaded into the VLNPs during the reassociation of the particles b. Table DOX) of 21.75 ± 0.80%, while 946 DOX molecules were packaged in each FA-N-tHBcAg VLNP with a loading efficiency (LEDOX) of 12.90 ± 0.50%. Technically, conjugation is simpler and can be performed more easily with a high coupling efficacy. However, the cytotoxicity of the dual conjugated VLNPs was lower than that of the pH-responsive VLNPs. This could be due to the fact that FA-tHBcAg-DOX VLNPs enter the cancer cells via FR-mediated endocytosis, and the drug can be released when the particles enter endosomes and are degraded. Therefore, these dual conjugated VLNPs are not pH-responsive. On the other hand, the pH-responsive VLNPs enter the cancer cells via FR-mediated endocytosis, and the encapsulated drug is released at tumour sites in a controlled manner, resulting in a higher therapeutic efficiency. For the dual conjugated drug delivery system, the number of DOX molecules conjugated to tHBcAg VLNPs was significantly higher compared to DOX loaded in the pH-responsive tHBcAg VLNPs. Approximately 1600 DOX molecules were conjugated to each FA-tHBcAg VLNP with a conjugation efficiency , as described by Tan et al. [The n et al. . The tHBn et al.  with somn et al. . 2HPO4/NaH2PO4, pH 7.4), and the FA conjugated VLNPs were separated by sucrose density gradient  as explained by Biabanikhankahdani et al. [The carboxylic acid groups of FA were activated by Sulfo-NHS and EDC, according to the method described by Biabanikhankahdani et al. . Then, ti et al. . The fra360) of FA-conjugated tHBcAg VLNPs was measured using a NanoDropTM 1000 spectrophotometer  at room temperature. The conjugated FA was quantified using an extinction coefficient of 5312 mol−1 cm−1, as described by Ren et al. [FA) and the number of FA (NFA) molecules conjugated to each nanoparticle were calculated using Equations (1) and (2), respectively. Absorbance at 360 nm , colorectal cancer cell line (HT29), and normal cell lines (3T3 and CCD-112) were obtained from the American Type Culture Collection (ATCC). HeLa and HT29 cell lines were grown in FA-deficient GIBCO RPMI1640 medium  while the 3T3 and CCD-112 cells were cultured continuously in DMEM and EMEM media, respectively , containing heat-inactivated fetal bovine serum  as a monolayer. The cells were kept in a humidified atmosphere of 95% air and 5% CO5 cell/mL) were sub-cultured in a six-well plate as described by Biabanikhankahdani et al. [In order to evaluate the internalisation property of FA-conjugated tHBcAg VLNPs into HeLa cells, anti-tHBcAg serum was used to detect the internalised tHBcAg particles. The cells  were then added. The mixture was stirred for further 16 h at 4 °C and then applied on a sucrose density gradient (8%–40%) to purify DOX-conjugated tHBcAg VLNPs. The amount of DOX in each fraction (400 µL per fraction) was measured at A495, and the amount of protein was determined with the Bradford assay [The carboxylate groups of the tHBcAg VLNPs were activated by EDC and Sulfo-NHS; then, the activated tHBcAg VLNPs were combined with DOX. tHBcAg VLNPs in 100 mM sodium phosphate buffer (pH 6.0) were incubated with freshly prepared solution of Sulfo-NHS (2000 M in excess) and EDC (1000 M in excess) at room temperature for 3 h with gentle stirring. After adjusting the pH of solution to 7.4, DOX•HCl (2000 M in excess) and DMSO (20% rd assay .−1 M−1) and A495 value, as described by Zeng et al. [DOX), and the number of conjugated DOX molecules to each nanoparticle (NDOX) were calculated with Equations (3) and (4) as follows:Protein fractions containing the highest amount of DOX-conjugated VLNPs were dialysed against 25 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl  at 4 °C, and concentrated with VIVASPIN 6. The amount of DOX was quantified based on its extinction coefficient  uranyl acetate at room temperature for 5 min and viewed under a Hitachi transmission electron microscope .tHBcAg VLNPs and derivatives were absorbed onto carbon-coated grids. Then, the grids were negatively stained with 2% (5 cells per well), as described by Biabanikhankahdani et al. [490 using a microplate reader . Cancer and normal cells were cultured in six-well plates , HT29 (2.0 × 105 cell/mL), 3T3 (1.0 × 105 cell/mL) and CCD-112 (1.0 × 105 cell/mL) cells were sub-cultured in six well plates. The old medium was then exchanged with 1 mL fresh medium containing FA-tHBcAg-DOX VLNPs, tHBcAg-DOX VLNPs, and free DOX with the equal DOX concentration (5 µg/mL), as described by Biabanikhankahdani et al. [480 nm and Em535 nm). Cellular uptake of FA-tHBcAg-DOX VLNPs, tHBcAg-DOX VLNPs, and free DOX was evaluated using live cell imaging. HeLa  and normal cells  were cultured in 96-well plates . Then, different concentrations of DOX formulations and free DOX (0.01–10 µM) in fresh medium were added to the cells. UV-absorbance at 570 nm was measured using the Uquant Elisa plate reader . The cytotoxicity of tHBcAg VLNPs towards these cells served as a negative control. In summary, this study demonstrated the in vitro efficacy of a dual conjugated drug delivery system derived from tHBcAg VLNPs for anticancer therapy. The tHBcAg VLNPs conjugated with DOX and FA significantly enhanced the cellular uptake and the amount of DOX in cancer HeLa and HT29 cells, leading to enhanced anti-tumour effects. The fabrication of this system is simple, and it has the potential to be easily modified for other combinatorial drug deliveries. Then, these dual bioconjugated VLNPs were compared with pH-responsive VLNPs. Both drug delivery systems have their advantages and limitations, depending on different factors such as the size, charge, molecular weight and solubility of the desired drug.


Many factors from the oocyte/sperm or the process of fertilization may affect the zygote formation. The zygote score (Z-score) describes the quality of a human zygote based on its pronuclear morphology, nucleolar precursor bodies, and alignment of polar bodies, and it can be used in the selection process at the zygote stage for embryo transfer or cryopreservation.The aim of this retrospective cohort study was to investigate the relationship between different controlled ovarian stimulation (COS) protocols and the zygote score (Z-score) and to assess the feasibility of the Z-score for predicting embryo survival in the GnRH-antagonist (GnRH-ant) protocol.It is a retrospective, single-center cohort study. A total of 3,826 zygotes with normal fertilization were analyzed from 744 in vitro fertilization /intra-cytoplasmic sperm injection (IVF/ICSI) cycles  between Jan 2010 and April 2014 in the IVF unit of Chang-Gung Memorial Hospital Kaohsiung Medical Center.The Z-score distribution differed significantly between these two protocols. The overall Z-score was poorer for zygotes from GnRH-ant cycles (p<0.05). Univariate and multivariate analyses indicated the type of COS protocol is one of the main determinants of Z-score grading. Our study found good-quality day 3 embryo/blastocyst formation and the cumulative embryo survival rate were correlated with the Z-score but not the COS protocol. With the GnRH-ant protocol, the number of Z1 in the transferred cohort embryos was significantly correlated with the clinical pregnancy rate  and live birth rate . This correlation was not seen with the long protocol.The Z-score distribution for the GnRH antagonist cycles was poorer than that of the long protocol, but the Z-score system is a valuable parameter for predicting embryo viability in the GnRH-ant protocol, providing a strong correlation with the clinical pregnancy rate and live birth rate. Clinicians have used the GnRH-ant protocol for assisted reproduction in recent decades as an alternative to the GnRH agonist (GnRH-ag) protocol –3. RecenThe zygote score (Z-score) is an established scoring system based on the pronuclear morphology, nucleolar precursor bodies, and alignment of polar bodies in the human zygote at approximately 16 to 18 h after insemination . ScoringThe present retrospective study examined a cohort of embryos derived from patients undergoing in vitro fertilization/intra-cytoplasmic sperm injection (IVF/ICSI). The aim of this study was to determine the relationship between the different controlled ovarian stimulation (COS) protocols and the Z- score and to determine the feasibility of using the Z-score to predict embryo survival in the GnRH-ant protocol.This retrospective study included 3,826 zygotes with normal fertilization from 643 women and followed 744 IVF/ICSI cycles at the Chang-Gung Memorial Hospital Kaohsiung Medical Center between January 1, 2010 and April 30, 2014. All of the women had cycles from COS with a luteal phase down-regulated protocol (long protocol) or a GnRH-ant protocol, and all couples had at least one zygote with two pronuclear (PN) formations after insemination/ICSI at 16 to 18 h. All cases with unsuccessful oocyte retrieval or polyspermic zygotes were excluded. Women were excluded if they underwent COS with natural cycles or clomiphene using an ultra-long protocol or a short protocol. All charts were retrospectively reviewed by one physician. The study was approved by an appropriately constituted the Institutional Review Board of the Ethics Committee of Chang Gung Memorial Hospital  and Institutional Review Board waived the need for consent. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.COS, oocyte retrieval, embryo culture, and embryo transfer were performed according to our previously described protocols , 19. TheFor PN scoring, zygotes were divided into 4 categories (Z1-Z4) based on 3 major features: size and location of the nuclei, appearance of the cytoplasm, and numbers, sizes, and distribution patterns of nucleolar precursor bodies within the nuclei. ) Z1 zygoTM medium (Scandinavian IVF Science) and on days 3 to 5 in G2 TM medium (Scandinavian IVF Science). Veeck’s morphological grading system [i.e. embryo arrest). In the analysis of embryo arrest, data from patients who received a day-2, day-3, day-4, day-5 or day-6 embryo transfer were censored after the time of transfer (loss due to transfer) or cryopreservation. Luteal phase support continued until the day pregnancy was confirmed by detection of hCG in the urine. If conception occurred, micronized progesterone supplementation was provided for an additional 4 weeks. Clinical pregnancy was defined as the presence of an intrauterine gestational sac with positive cardiac movement on ultrasound [Embryos were cultured on days 1 to 3 in G1 g system  was adoptrasound . The liv2 test or Fisher’s exact test was used to compare categorical variables. A Pearson correlation coefficient (r) was calculated for correlations. All statistical analyses were performed with SPSS, version 17.0 . All p values were two-sided, and a p value less than 0.05 was considered statistically significant. Logistic regression analysis was used to determine the significance of factors associated with a Z-score of 1 (Z1). Multivariate logistic regression analysis was then performed to further examine factors associated with Z1, considering the following variables: female age, male age, ICSI or IVF, etiology of infertility, body mass index, COS protocol, days of stimulation, FSH dosage, E2 level on the day of hCG detection, E2 level per oocyte, P4 level, maturity of oocyte, and number of same-cohort oocytes. Cumulative survival rates were calculated by life-table analysis using the Kaplan–Meier product limit procedure at each day. The differences between groups with different Z-scores were assessed using the log-rank test. A p-value less than 0.05 was considered significant [Continuous data are given as the mean ± standard deviation (SD). The Mann-Whitney rank sum test was used to compare continuous data, and the χnificant .During the period, 744 fresh cycles from 643 women were included, which consisted of 352 long protocol cycles and 392 GnRH-ant protocol cycles. p < 0.05) and shifted toward poorer Z-scores in the GnRH-ant protocol.A total of 3,826 zygotes with normal fertilization produced from 744 fresh cycles following IVF/ICSI were examined. We performed fertilization check using the Z-score, a system that has been used to evaluate fertilization in our center since 2001. p < 0.001), oocyte maturity (p < 0.001), progesterone on the day of hCG detection (p < 0.001), E2 concentration per oocyte (p = 0.028), total FSH dosage (p = 0.025), and duration of FSH stimulation (p = 0.028).In this retrospective and observational study, we did not perform statistical comparisons between cycles from the different protocols due to the risk of selection bias. Instead, we observed the cohort zygotes from the two protocols and analyzed potential factors that may influence the zygote score. Our previous study indicated good (Z-1) zygote had higher implantation potential.We furthep < 0.001), number of cohort 2PN (p < 0.001), and COS protocol used (p < 0.001). The observation of significantly fewer Z1 zygotes from the GnRH-ant protocol is compatible with our clinical observations.We then used multivariable logistic regression analysis to identify factors associated with a score of Z1 . These rvs. 370(65.3%), Z2: 174(22.4%) vs. 208(23.7%), Z3: 13(4.1%) vs. 21(5.2%), Z4: 0(0%) vs. 1(1%); p = 0.696) (vs. 198(35%), Z2:123(16%) vs. 194(22%), Z3: 9(3%) vs. 45(11%), Z4:0(0%) vs. 2(3%); p = 0.066)   . An anal= 0.066) . Further= 0.066) . The ove= 0.370) . As expep <0.05; .vs. 94 ± 1%, p < 0.001)   , Z2 zygo< 0.001) , and Z4 = 0.022) , but not= 0.615) .Thus, our study found that good-quality day 3 embryo/blastocyst formation and the cumulative survival rate were correlated with the Z-score but not the COS protocol.r = 0.976; p = 0.088) or live birth rate . For the GnRH-ant protocol, the number of Z1 in the transferred cohort embryos was significantly correlated with the clinical pregnancy rate  and live birth rate .To our knowledge, the current study is the first to report the effect of different COS protocols on zygote morphology and the feasibility of using the Z-score to predict embryo survival in the GnRH-ant protocol.Zygote formation follows the dramatic reorganization of sperm chromatin by many factors stored within the oocyte , 26. TheMany factors from the oocyte/sperm or the process of fertilization may affect the Z-score. Our IVF laboratory adopted a modified Scott scoring system beginning in 2001 to monitor fertilization. Our previous report  indicated that the Z‐score is an additional criterion that can be used to select embryos for extended culture and is useful when selecting embryos for transfer. However, the distribution of Z‐scores changes significantly when the GnRH-ant protocol is used for COS. In particular, we found that the Z-scores from the GnRH-ant protocol were generally poorer than those from the long protocol. Significantly fewer Z1 zygotes resulted from the GnRH-ant protocol, which is compatible with our clinical observations.et al. [Some possible explanation for the differences in the Z-score between the COS protocols involves the different endocrine profiles during follicular growth. First, the long protocol induces profound suppression and leads to simultaneous maturation of antral follicles , 31, 32.et al. , 36, butet al. , but onlet al. –40. The r = 0.971; p = 0.029). However, this correlation was not observed for the long protocol. A possible explanation for the Z-score having better predictive value in the GnRH-ant protocol is that zygotes from the GnRH-ant protocol were taken from a more natural pattern of follicle recruitment, while the long protocol only improved the zygotes’ morphology without changing the survival potential.Although more mature oocytes/Z-1 zygotes were produced from long protocol than GnRH-ant possibly because of better endocrine profiles and no effect of GnRH antagonist; however, the overall Z-1 zygotes from long protocol had poorer survival and not correlated with clinical pregnancy. Our results suggested that long protocol only improved morphological maturity of oocytes and zygotes’ morphology without changing the survival potential of oocyte. Some Z1 zygotes from long protocol may originated from immature oocytes with poorer potential and therefore had poorer pregnancy outcomes than Z1 zygotes from GnRH-ant protocol. The findings may also explain why Z-score could not be consistently verified in previous studies which used long protocol. There has been debate regarding the use of the Z-score for predicting outcomes following assisted reproduction , 41–49. The present study has some limitations. First, this retrospective study has risk of selection bias. A matched control group might improve the quality of the findings. Second, the relationship between pregnancy outcomes and Z-score for single embryo transfer requires further investigation. A mean of approximately two embryos were transferred to most patients in this study, so single-embryo outcomes could not be compared with the Z-score. Third, the timing of assessment is critical, as pronuclear development is a dynamic process. Thus, determination of the Z-score from a single light microscopy observation should be used with caution and only in conjunction with other methods of evaluation .In conclusion, the Z-score distribution was poorer for the GnRH-ant protocol than the long protocol. This may be because these protocols have different effects on the endocrine profile or GnRH receptors. Additionally, the Z-score is a more feasible parameter for predicting embryo viability in IVF/ICSI with the GnRH-ant protocol than the long protocol.S1 FigZygote scoring system of Scott et al. Z1 zygot(TIF)Click here for additional data file.


Huntington's disease (HD) is one of several neurodegenerative disorders that have been associated with metabolic alterations. Changes in Insulin Growth Factor 1 (IGF-1) and/or insulin input to the brain may underlie or contribute to the progress of neurodegenerative processes. Here, we investigated the association over time between changes in plasma levels of IGF-1 and insulin and the cognitive decline in HD patients.We conducted a multicentric cohort study in 156 patients with genetically documented HD aged from 22 to 80 years. Among them, 146 patients were assessed at least twice with a follow-up of 3.5 ± 1.8 years. We assessed their cognitive decline using the Unified Huntington’s Disease Rating Scale, and their IGF-1 and insulin plasmatic levels, at baseline and once a year during the follow-up. Associations were evaluated using a mixed-effect linear model.In the cross-sectional analysis at baseline, higher levels of IGF-1 and insulin were associated with lower cognitive scores and thus with a higher degree of cognitive impairment. In the longitudinal analysis, the decrease of all cognitive scores, except the Stroop interference, was associated with the IGF-1 level over time but not of insulin.IGF-1 levels, unlike insulin, predict the decline of cognitive function in HD. The central nervous system plays a key role in the regulation of neuroendocrine axes and, in turn, the released neurohormones participate to the activity of different areas of the brain. On the one hand, Insulin-like growth factor-1 (IGF-1), the main effector of the somatotropic axis, has a wide range of actions in both the central and peripheral nervous system, demonstrated in various experimental models in animals and humans . MoreoveHuntington’s disease (HD) is an autosomal, dominantly inherited, progressive neurodegenerative disorder characterized by involuntary movements, especially chorea, cognitive decline, behavioural, and psychiatric disturbances, all leading to functional disability. This rare and fatal disease is caused by an abnormal elongation of a CAG repeat sequence in the gene located on the short arm of chromosome 4 and encoding the protein huntingtin [Studies in animal models and human patients found evidence that endocrine and metabolic alterations accompany the progression of HD. On the one hand, decreased concentrations of the IGF binding complex and the significantly higher basal plasma levels of IGF-1 in HD patients than in controls support the hypothesis of a resistance to IGF-1 in patients with HD . In addiTherefore, we aim to assess the association between changes in plasma levels of IGF-1 and insulin and the cognitive decline in HD patients over time, in a prospective multicentric cohort study.Htt gene (> 36 CAG repeats in mHtt) and aware of their genetic status. They had neither other neurological conditions nor long-term experimental treatment (e.g. cell transplantation).We report a longitudinal prospective study of manifest Huntington’s disease patients from the Predictive Biomarkers for Huntington’s disease protocol (NCT01412125), which was approved by the ethics committee of Henri Mondor Hospital  in accordance with EU and French bioethics laws. All patients gave written informed consent. They were heterozygous for the Data were collected from 2003 to 2009, at six centers from the French Speaking Huntington’s Disease Group , and centralized at the National Reference Centre for Huntington’s disease at Créteil.One hundred fifty six patients were assessed at baseline. One hundred and fourty six patients 94%) had at least two clinical and biological assessments so they were included in the longitudinal analysis. They were assessed once a year during the follow-up in each center. The maximum follow-up duration was of 7 years with a mean ± SD of 3.5 ± 1.8 years. The mean number of visits was 2.13 ± 1.11 . At each assessment, scores were given on the motor, cognitive, behavioural, and functional sections of the French version of the Unified Huntington’s Disease Rating Scale (UHDRS). The cognitive function was assessed using the 3 standardized tests of the UHDRS: the Stroop Interference Test, the Symbol Digit Modalities Test (SDMT), and the verbal fluency test  [4% had atIn addition, the patients were interviewed about medical history and completed a questionnaire on past and current symptoms. Medication use of the participants was recorded, including antipsychotics, antidepressants and tranquillizers.Etablissement Français du Sang at Avicennes. All samples were tested in a single run using a single reagent lot at the Département de Biochimie-Pharmaco-Toxicologie at Henri Mondor. Kits were used according to the manufacturers’ instructions. For IGF-I and insulin-like growth factor-binding protein 3 (IGFBP-3), we used the Isys kit . For insulin, we used RIA kits using Glucose Analyzer 2 .At baseline and at each follow-up clinical examination, a blood sample from each patient was collected in the morning after an overnight fast, then centralized, centrifuged and stored at -80°C at the Linear regression was performed at baseline to evaluate the associations between serum levels of IGF-1 or insulin and UHDRS cognitive scores.In the longitudinal analysis, the individual annual slope of cognitive assessment was estimated for each task using a mixed-effect linear model with ‘‘patient” as a covariance estimate. This model takes into account the repeated measures at several time points and so the intra-individual variability. Then, we ran univariate analyses with each cognitive score as a dependent variable, and IGF-1 or insulin as independent variables in separate models. The multivariate analyses were done to adjust our models on the following potential confounding variables: age, age at onset, gender, body mass index (BMI), CAG, antipsychotics treatment, and level of education. In addition, models on IGF-1 were further adjusted for its main binding protein IGFBP-3 that regulates its activity.β regression coefficients for the linear models. A two-sided p-value ≤ 0.05 was considered statistically significant. Statistical analyses were conducted using SAS version 9.0 .Results are presented as means with their standard deviation (SD) and as mean regression slopes and their standard error (SE) or as p = 0.06; ß Stroop color naming, -3.50, p = 0.04; ß Stroop interference, -5.42, p = 0.02; and ß SDMT, -3.58, p = 0.02; Insulin: ß Stroop word reading, -0.19, p = 0.06; ß Stroop color naming, -0.19, p = 0.08; ß Stroop interference, -0.22, p = 0.03; and ß verbal fluency test, -0.25, p = 0.003).In the cross-sectional analysis, cognitive scores at baseline showed moderate impairment compared with published reference values . Both plAll cognitive scores declined significantly during the follow-up . ChangesIn this prospective multicentric cohort study of HD patients followed for 3.5 ± 1.8 years, we found that higher baseline plasma levels of IGF-1 and insulin were associated with greater impairment in executive functions and attention as assessed by the cognitive part of the UHDRS. These significant associations were independent from potential confounders including age, age at onset, number of CAG repeats, sex, BMI, level of education, antipsychotics treatment, and plasma IGFBP-3 for IGF-1. In the longitudinal analysis, we showed that the decline of the UHDRS cognitive scores was statistically significant over time. The worsening of all cognitive scores, except Stroop interference, was inversely associated to plasma IGF-1 levels over time, unlike insulin.Our results confirm that the elevated plasma levels of IGF-1 observed in earlier studies of HD  are assoIGF-1 is commonly described as not only involved in brain growth, development and myelination, but also in brain plasticity and neurogenesis . Normal Since the early 1990s, intervention studies in animal models have shown neuroprotective effects of IGF-1 in different models of injury and insults to the brain, administered through different routes, for example, directly through intracerebroventricular administration  or peripBesides predicting the cognitive decline in HD, IGF-1 plasma levels over time are inversely associated to the cognitive functions, independently of other confounding factors such as age. Thus, IGF-1 is a potential candidate as a biomarker for predicting or evaluating the progress of the disease. However, its relevance for clinical application requires additional well-defined criteria and thus further investigation.As for insulin, our cross-sectional results show that clinical hyperinsulinism is negatively associated to cognition in Huntington’s disease. Our results appear to be in line with many studies which show that hyperinsulinemia and insulin resistance are risk factors for developing dementia and affect the initiation of neuritic plaques formation ,25. In fHowever, despite the structural homology between IGF-1 and insulin, both at molecule and receptor levels, and despite their functional interrelationship, the insulin levels over the follow-up period did not show statistically significant associations with any of the cognitive scores changes. Thus, the control of insulin levels may not be a therapeutic target in HD. This finding opposes to other findings of neurothrophic properties of insulin infusion ,29 and aTo our knowledge, this is the first study providing a long-term follow-up (up to seven years) of 146 HD patients assessing their clinical and plasmatic IGF-1 and insulin levels. Our study gives a piece of information on the impact of IGF-1 and insulin on cognition in HD. IGF-1 levels over time predict and are associated to the decline of cognitive function, unlike insulin. The temporality between IGF-1 resistance and neurodegeneration in HD remains to be explored. In addition, it would be interesting to further investigate the role of the downstream PIK3/Akt pathway in resistance to IGF-1 on one hand and in the cognitive decline in HD on the other hand.